US11535684B2 - Chitosan derivatives and methods for preparing the same - Google Patents
Chitosan derivatives and methods for preparing the same Download PDFInfo
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- US11535684B2 US11535684B2 US17/270,237 US201917270237A US11535684B2 US 11535684 B2 US11535684 B2 US 11535684B2 US 201917270237 A US201917270237 A US 201917270237A US 11535684 B2 US11535684 B2 US 11535684B2
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/722—Chitin, chitosan
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Definitions
- the invention relates to methods of preparing derivatives of chitosan.
- the invention further relates to chitosan derivatives.
- Chitosan is a linear polysaccharide derived from chitin, an abundant natural polymer, found in the exoskeleton of crustaceans, insects and anthropoids and in fungal cell wall. It is mostly derived from marine sources such as shrimp, lobster and crab shells. Chitosan is the deacetylated form of chitin. Full deacetylation of chitin results in chitosan with a degree of deacetylation of 100%, and has the general formula
- Chitosan is a commercial product that has versatile applications in several industries. This includes its use as a hydrating compound in cosmetics, as foods preservative, as stabilizer, as food supplement, environmental friendly packaging and dietary fiber, wound healing agent in pharmaceutical application, as drug delivery system as well as several other promising applications (M. Kong, et al., Int. J. Food Microbiol., 144 (2010) 51-63).
- Trimethylchitosan (N,N,N-trimethylchitosan, TMC) is a chitosan derivative with number of interesting biological applications and the advantage of high solubility independent of pH and strong antimicrobial effect and potential as absorption enhancer. Although unmodified chitosan can also have antimicrobial effect and function as absorption enhancer its effectiveness is limited to low pH ( ⁇ 6) due to the lack of solubility.
- Trimethylchitosan is typically synthesized by reacting chitosan with a methylating agent (typically Methyl iodide (MeI) or dimethyl sulfonate (DMS) in the presence of a base (typically NaOH). Chitosan is insoluble in aqueous solutions under such basic conditions so the reaction will typically be performed in highly polar organic solvent such as N-methyl-2-pyrrolidone (NMP).
- NMP N-methyl-2-pyrrolidone
- N,N,N-trimethylation 86% N,N,N-trimethylation was obtained with very low O-methylation.
- Another reported method is reductive alkylation (N,N-dimethylation) in the first step and methylation with a MeI. This two-step procedure resulted in 73% N,N,N-trimethylation with low O-methylation (Verheul, R. J., et al. Biomaterials 29 (2008) 3642-3649).
- Benediktsdóttir et al. have reported synthesis of N,N,N-trimethylchitosan from TBDMS chitosan (B. E. Benediktsdóttir et al., Carbohyd.
- the present invention provides a simple and efficient procedure for the synthesizing trialkylated chitosan, e.g. trimethylchitosan, with very high degree of N,N,N-trialkylation and very low O-alkylation.
- the procedure is based on dissolving the reagent chitosan in acid and the use of a base and a moderate excess of the alkylating agent in a polar water-miscible solvent to yield the desired N,N,N-trialkylated compound in a single alkylation step. No isolation or purification of an intermediate, partially N-alkylated or N,N -dialkylated compound is required.
- the same approach can be used for to prepare N-alkylated chitosan derivatives with mixed alkylation by using more than one alkylation agent.
- the invention in an aspect relates to a method of the preparation of N-alkylated chitosan the method comprising steps of
- the invention relates to a method for the preparation of a compound of Formula (I)
- n is an integer greater than or equal to 3
- W is N-acetyl
- each R 1 substituent may be the same or different and is independently selected from H, CH 3 , and —(CH 2 ) a —CH 3 , wherein a is from 1 to 11, or
- each R 1 substituent is independently selected from H, CH 3 , and —(CH 2 ) a —CH 3 , wherein a is from 1 to 11, and wherein R 2 is selected from:
- i is an integer from 1-200, preferably 1-50, more preferably 1-10, and R 3 is selected from H, —(CH 2 ) j —CH 3 and —CO—(CH 2 ) j —CH 3 , wherein j is 0, 1, 2, 3, 4 or 5; and
- R is H in at least 75 molar % of the total number of positions, and when it is not H, R is the same as either amino substituent R 1 or R 2 on substituent W;
- each S 1 substituent is independently selected from CH 3 , and —(CH 2 ) a —CH 3 , wherein a is from 1 to 11, or wherein S 1 is selected from:
- i is an integer from 1-200, preferably 1-50, more preferably 1-10, and S 2 is selected from H, —(CH 2 ) j —CH 3 and —CO—(CH 2 ) j —CH 3 , wherein j is 0, 1, 2, 3, 4 or 5
- chitosan dry salt is initially prepared by dissolving chitosan in an acidic solution and then precipitating the chitosan as a salt. The chitosan can then be dissolved in water in the salt form without further addition of acid.
- the base and alkylating agent can be added in any order or concomitantly. Accordingly, in some embodiments, the alkylating agent is added prior to adding the base. In some embodiments, the base is added prior to adding the alkylating agent. In some embodiments, the base and the alkylating agent are added concomitantly.
- the reaction to prepare N-alkylated chitosan derivatives can be repeated to obtain very high degree of trialkylation with minimal O-alkylation.
- the product N-alkylated chitosan is subjected to a second round of N-alkylation using the same reagents as in the first round of alkylation.
- the method of the invention can further comprise isolating the product N-alkylated compound of formula (I) and subjecting the thus isolated product to a second round of treatment according to step (b), wherein in the second round of treatment, the same or different alkylation reagent is used, compared with the alkylation reagent used to isolate the isolated N-alkylated compound.
- the second round of treatment a further product N-alkylated compound of formula (I) with a higher degree of N,N,N-trialkylation is obtained.
- the intermediate compound (the product of the first alkylation reaction) is dissolved prior to performing the second alkylating step.
- the dissolving can involve the addition of acid.
- the invention also relates to N-alkylated chitosan compounds.
- the invention relates to compounds having the formula
- each R 1 substituent may independently be selected from H, CH 3 , and —(CH2) a —CH 3 , wherein a is from 1 to 11, and wherein R 2 may be selected from:
- i is an integer from 1-200, preferably 1-50, more preferably 1-10, and R 3 is selected from H, —(CH 2 ) j —CH 3 and —CO—(CH 2 ) j —CH 3 , wherein j is 0, 1, 2, 3, 4 or 5,
- R is H in at least 75% of the total number of positions, and when it is not H, R is the same as amino substituent R 1 or R 2 in W;
- the N,N,N-alkylated compound can be a salt having any suitable counterion.
- the N,N,N-alkylated compound is a salt that has a counterion that is selected from Cl ⁇ , Br ⁇ and CH 3 OSO 2 O ⁇ .
- R 1 is not H in all monomer units, i.e., not all of the monomer units contain H in the R 1 position. In some embodiments, R 1 is not H in at least 5% of monomer units, in at least 10% of monomer units or at least 20% of monomer units.
- the compounds described herein can be used in treatment options known for chitosan, including their use as bacteriocidal agents, as fungicidal agents, as mucoadhesives, as haemostatic agents, as tissue regeneration agents, as wound healing agent or as bone regeneration agents.
- FIG. 1 shows H 1 -NMR spectrum of the product from Example 1—the solvent was D 2 O+acetic acid-d 4
- FIG. 2 shows H 1 -NMR spectrum of product from Example 2—the solvent is D 2 O+acetic acid-d 4
- FIG. 3 shows H 1 -NMR spectrum of product from Example 3—the solvent is D 2 O+acetic acid-d 4 . Dialyzed sample of the material.
- FIG. 4 shows H 1 -NMR spectrum of product from Example 4—the solvent is D 2 O. Dialyzed sample of the material.
- FIG. 5 shows H-NMR spectrum of the product from Example 5. Sample submitted in 1% acetic acid-d 4 in D 2 O.
- FIG. 6 shows H-NMR spectrum of the product from Example 6. Sample submitted in 1% acetic acid d 4 in D 2 O.
- FIG. 7 shows 1 H-NMR spectrum of TMC chitosan.
- the invention relates to novel N-alkylated chitosan derivatives, as well as a method for preparing such N-substituted chitosan derivatives.
- the procedure in general can be carried out only a relatively small excess of the alkylating agent, much less excess than in the original procedure disclosed for the preparation of N-methylated chitosan (700% excess of methylating agent).
- the process of preparing N-alkylated chitosan can also be carried out in a single alkylation step, not requiring the isolation of intermediate compounds. This is a distinct advantage over prior art methods of preparing e.g. trimethylchitosan, which require the isolation of intermediate dimethylchitosan.
- the resulting compound can be produced with minimal O-alkylation, which is a major side product in the preparation of trimethylchitosan.
- Chitosan is the deacetylated form of chitin.
- chitosan When fully deacetylated, chitosan is a polymer of 2-amino-2-deoxy-D-glucose linked by ⁇ -(1-4) bonds.
- chitosan When partically (i.e., not 100%) deacetylated, chitosan is a copolymer of N-acetyl-2-amino-2-deoxygl-D-glucose and 2-amino-2-deoxy-D-glucose.
- Chitosan is normally obtained by deacetylation of chitin. However, since it is difficult to obtain fully deacetylated chitin, chitosan normally contains some N-acetylation.
- the method and compounds described herein can contain a portion of N-acetylated material; in other words, the chitosan as described herein can be either completely or partially deacetylated.
- the acetylated portion of the starting material will not react with the methylation agents described herein and will therefore remain silent in the synthetic process of the N-substituted chitosan derivatives.
- Chitosan has versatile applications, such as in medicine, food agriculture, cosmetics, nutraceutical, environment friendly packaging and others.
- Chitosan has been postulated to be useful in the treatment of a number of human conditions.
- the compound is useful in medical treatment since it has high degree of biocompatibility and is biodegradable.
- Chitosan has bacteriocidal activity and is well adsorbed.
- chitosan is useful in wound treatment and can be used in e.q. wound dressings and to dampen immunologic mediators such as IL-8, prostaglandin E, IL-1 beta and others.
- Chitosan can be formulated using known methods, e.g., as beads, gels, microparticles, nanoparticles, nanofibers and as scaffold to support normal (healthy) tissue. Chitosan is thus used in 3D scaffolds such as gels and sponges.
- haemostatic activity and antifungal activity chitosan can be used in chitosan sponges (a type of porous dressing) that have exudative properties in wound treatment in addition as aiding in tissue regeneration, including bone regeneration, thus promoting bone regeneration.
- Chitosan has a range of other uses, such as in waste water treatment, in heavy metal removal, as focculating agent, as an antioxidant and for skin protection in cosmetic compositions.
- the compounds described herein are contemplated to be useful in all known uses of chitosan, including but not limited to the foregoing.
- chitosan Despite its large range of useful properties, chitosan possesses poor aqueous solubility, which gives rise to different chemical modifications of chitosan in order to obtain better water solubility and improve biological properties.
- Two of the most notable derivatives are N,N,N-trimethyl chitosan (TMC) and carboxymethyl chitosan (CMC).
- TMC is soluble in aqueous solutions and it has shown that this chitosan derivative can be remarkably more active against bacterial infections than unmodified chitosan and may also have significant value as an absorption enhancer for drug delivery applications (Benediktsdóttir et al. 2014).
- N,N,N-trimethylchitosan is produced by N-methylation of chitosan.
- alkylating reagents like methyl iodide (MeI) have been used for this purpose.
- MeI methyl iodide
- the resulting product possesses a permanent positive charge and improved aqueous solubility in a wide pH range compared to native chitosan (Benediktsdóttir et al. 2014). With these improved properties, TMC is a good candidate for a variety of uses, including in drug delivery systems and gene delivery at physiological pH, as an antibacterial compound or in the treatment in various human conditions.
- the present invention makes it feasible to prepare N,N,N-trimethylchitosan in high yield with minimal O-methylation, thereby providing a distinct advantage over prior art protocols.
- the invention also relates to a novel method of preparing alkylated chitosan.
- the invention further provides novel N-alkylated chitosan derivatives.
- These derivatives can be used in methods and processes known for chitosan, for example as antibacterial, antifungal or antiviral agents, in haemostasis, in drug delivery systems and gene delivery, in the treatment of human conditions, tissue and bone regeneration, in wound dressings, and as wound-healing agents.
- the compounds disclosed herein can also be used to enhance drug adsorption.
- N-alkylated chitosan derivatives described herein are in general water soluble and therefore particularly useful in a range of applications.
- the insolubility of chitosan is overcome by the N-alkylation, while retaining the beneficial characteristics of the chitosan compound.
- chitosan should be (thermodynamically) insoluble under basic or neutral conditions.
- a key feature of the method is the prior dissolution of the chitosan by the addition of acid (thereby forming a chitosan salt) before adding the alkylating agent(s).
- the reagents and product remain largely in solution.
- the alkylation proceeds in a single reaction; isolation of intermediate partially alkylated compounds is not required or needed.
- Chitosan is a deacetylated form of chitin.
- chitosan is partially deacetylated, which means that the chitosan is a heterogeneous mixture of oligomers containing different amount of N-acetylation.
- the chitosan compound used as reagent in the present invention can be from 1 to 60% N-acetylated, such as from 1 to 50% N-acetylated, from 1 to 40% N-acetylated, from 1 to 30% N-acetylated, from 1 to 20% N-acetylated, or from 1 to 10% N-acetylated (all percentages being molar %).
- the inventors have found that by treatment with acid prior to the addition of alkylating agent, the reagents are brought into solution, thereby increasing the yield of the subsequent alkylation step.
- the acid can be any mineral acid, such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, perchloric acid, nitric acid, or mixtures of such acids.
- the mineral acid is a monovalent acid.
- the acid can also be one or more organic acid, such as acetic acid, formic acid, oxalic acid, lactic acid, propionic acid, maleic acid, succinic acid, methanesulfonic, acid, p-toluenesulfonic acid.
- organic acid such as acetic acid, formic acid, oxalic acid, lactic acid, propionic acid, maleic acid, succinic acid, methanesulfonic, acid, p-toluenesulfonic acid.
- An alkylating agent and at least one base is subsequently added to the aqueous chitosan solution.
- the alkylating agent and base can be added concomitantly or in any order.
- the alkylating agent can be added prior to the base.
- the amount of base can further be from 0 to 5 equivalents relative to the number of equivalents of alkylating agents in the reaction, such as from 0 to 4 equivalents, from 0 to 3 equivalents, from 0 to 2 equivalents, or from 1 to 2 equivalents.
- the base can in principle be any base, such as for example sodium hydroxide. It can however be beneficial to use a mild or weak base.
- Useful bases in the reaction can for example be one or more of NaHCO 3 , Na 2 CO 3 , KHCO 3 , K 2 CO 3 , RbHCO 3 , Rb 2 CO 3 , CsHCO 3 , Cs 2 CO 3 , triethylamine, tripropylamine, tributylamine, triisopropylamine, triisobutylamine, N,N,-diisobutylmethylamine, triisopropanolamine, N,N-diisopropylethylamine, N,N,-diisopropyl aniline, 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU).
- Other weak bases known in the art are however also contemplated.
- a single base can be added, or a mixture of bases (e.g. a mixture of
- the procedure in general can be carried out only a relatively small excess of the alkylating agent.
- the alkylating agent is added in a stoichiometry that is at least 1:1 with respect to the equivalents of NH 2 in the starting chitosan compound.
- the alkylating agent in an amount of from 1 to 15 equivalents, from 1 to 12 equivalents, from 1 to 10 equivalents, from 1 to 9 equivalents, from 1 to 8 equivalents, from 1 to 6 equivalents, from 1 to 4 equivalents, or from 1 to 3 equivalents with respect to the equivalents of NH 2 in the starting chitosan compound.
- the degree of O-alkylation can be kept to a minimum in the reaction.
- the N-alkylated chitosan product of the reaction can have less than about 25% O-alkylation, less than about 20% O-alkylation, less than about 15% O-alkylation, less than about 10% O-alkylation, less than about 5% O-alkylation, less than about 2% O-alkylation, or less than about 1% O-alkylation (all being molar %).
- the reaction product is essentially free of O-alkylation.
- the method of the invention can thus further comprise a step of determining the degree of O-alkylation in the product.
- the O-alkylation as so determined can therefore preferably be found to be (molar %) less than about 25% O-alkylation, less than about 20% O-alkylation, less than about 15% O-alkylation, less than about 10% O-alkylation, less than about 5% O-alkylation, less than about 2% O-alkylation, or less than about 1% O-alkylation. In some embodiments, the O-alkylation is not detectable.
- the method can accordingly include a step of determining the degree of O-alkylation in the product(s), with the O-alkylation as so determined being as described in the foregoing.
- the solvent used in the method can comprise at least one water-miscible solvent.
- the solvent can be in a mixture with water.
- the starting material is dissolved in a a water-miscible solvent, such as a water miscible polar solvent, or a mixture of two or more such water miscible polar solvents.
- a water-miscible solvent such as a water miscible polar solvent, or a mixture of two or more such water miscible polar solvents.
- Exemplary solvents that are useful include N,N-Dimethylformamide (DMF), N-Methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), NMP and DMSO N,N-dimethylacetamide (DMAC), 1,4-dioxane, methanol, ethanol, tetrahydrofuran, acetonitrile.
- the solvent can include any one of these solvents, or mixtures of two or more of these solvents, optional
- the alkylation reaction can in general be carried out at any suitable temperature.
- the reaction can be carried out at a temperature that is preferably at least 20° C. It can be useful to carry out the reaction at higher temperatures, such as at least 30° C., at least 40° C., at least 50° C., at least 60° C., at least 80° C., at least 100° C., or at least 120° C. Temperatures above the boiling point of the solvent can be achieved by carrying out the reaction under pressure.
- the temperature of the reaction is in the range of 20° C. to 200° C., in the range of 20° C. to 150° C., in the range of 20° C. to 100° C., in the range of 40° C. to 70° C., or in the range of 50° C. to 60° C.
- catalysts known in the art include sodium iodide.
- the alkylating reagent in the second alkylation can also be different from the alkylating agent used in the first alkylation reaction. This has the advantage of being able to tailor the product obtained, both in terms of composition of the alkylating groups and also in terms of the degree of N-substitution with particular groups.
- a compound that contains a high degree of N-methylation and a smaller degree of alkylation with a longer alkyl group for example propyl or butyl
- the alkylation can be carried out in this manner using any combination of alkyl groups, varying the reaction conditions (such as amount of alkylating reagent) to achieve the degree and composition of N-alkylation that is desired.
- the alkylated chitosan derivatives disclosed herein can be useful for use as a medicament.
- the alkylated chitosan derivatives disclosed herein can be formulated using known formulation methods, including formulations as beads, gels, microparticles, nanoparticles, nanofibers and as tissue-supporting scaffolds.
- the chitosan derivatives can be used as bacteriocidal agents, as fungicidal agents, as mucoadhesives, as haemostatic agents, as tissue regeneration agents, as wound healing agent or as bone regeneration agents.
- the alkylated chitosan derivatives may also be formulated as tablets or capsules.
- the formulations can be adapted for non-invasive or invasive delivery.
- the compositions may be formulated for oral, topical, transmucosal, vaginal, ocular and rectal or inhalation delivery.
- the compositions may also, or alternatively, be formulated for injection.
- the compositions may further be formulated for immediate or sustained delivery, or the compositions may be formulated for delayed delivery.
- the formulations may include one or more pharmaceutically acceptable excipient.
- excipients generally can serve the role of providing the resulting composition with increased long-term stability, facilitate adsorption, enhance solubility, providing flowability or non-stick properties and also prevent degradation or aggregation over time.
- excipients include fillers, binders, disintegrants, coatings, sorbents, antiadherents, lubricants, glidants, preservatives, antioxidants, flavouring agents, sweeteners, colouring agents, solvents, buffering agents, chelating agents, viscosity imparting agents, surface active agents and humectants.
- novel N-alkylated compounds can also be used as excipients in pharmaceutical formulations (see Singh et al., Int J Pharm Sci Res 2 (2011) 2266-2277).
- the present invention also covers the exact terms, features, values and ranges etc. in case these terms, features, values and ranges etc. are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (i.e., “about 3” shall also cover exactly 3 or “substantially constant” shall also cover exactly constant).
- the degree of N,N,N-trimethylation (D-TM) of the free amino groups in the N-methylated chitosan product was 72% and N,N-dimethylation (D-DM) was 28% according to H 1 -NMR ( FIG. 1 ) and O-methylation was not detected.
- N,N,N-trimethylation (D-TM) in the N-methylated chitosan product was 68% and N,N-dimethylation (D-DM) was 32% according to H 1 -NMR ( FIG. 2 ) and O-methylation was not detected.
- the degree of N,N,N-trimethylation (D-TM) in the N-methylated chitosan product was 26% and N,N-dimethylation (D-DM) was 67% and N-monomethylation (D-MM) was 1% according to H 1 -NMR ( FIG. 4 ) and O-methylation was not detected.
- N-ethylation N-alkylation
- the degree of N,N,N-trialkylation (D-TA) in the N-ethylated chitosan product was 26% and the N,N-dialkylation (D-DA) was 74% according to H 1 -NMR ( FIG. 5 ).
- the degree of hydroxypropylation (D-HP) in the chitosan product was 76%, the degree of tertiary methylation (D-TeM) was 35% and the degree of quaternary methylation (D-QM) was 14% according to H 1 -NMR ( FIG. 6 ).
- the degree of tetriary methylation (D-TeM) was 57% and the degree of quaternary methylation (D-QM) was 37% according to H 1 -NMR.
- the degree of hydroxypropylation (D-HP) was 20%.
- D-TeM degree of tetriary methylation
- D-QM degree of quaternary methylation
- D-HP degree of hydroxypropylation
- D-TeM degree of tetriary methylation
- D-QM degree of quaternary methylation
- D-HP degree of hydroxypropylation
- the degree of N,N,N-trialkylation in the N-ethylated chitosan product was 43% and the N,N-dialkylation was 57% according to H 1 -NMR.
- the degree of N-ethylation was 32%.
- the degree of N,N,N-trimethylation in the N-methylated chitosan product was 60% and N,N-dimethylation was 30% and N-monomethylation was 10% according to H 1 -NMR and O-methylation was not detected.
- the degree of N,N,N-trimethylation in the N-methylated chitosan product was 67% and N,N-dimethylation was 30% and N-monomethylation was 3% according to H 1 -NMR and O-methylation was not detected.
- the degree of N,N,N-trimethylation in the N-methylated chitosan product was 42% and N,N-dimethylation was 56% and N-monomethylation was 5% according to H 1 -NMR and O-methylation was not detected.
- the degree of N,N,N-trimethylation in the N-methylated chitosan product was 92% and N,N-dimethylation was 8% according to H 1 -NMR and O-methylation was not detected.
- N,N-Diisopropylethylamine is added again. After 20 hours of heating and refluxing the reaction mixture, it is allowed to room temperature, the product is precipitated with 200 mL EtOH, and then filtered on sintered funnel. The solid is washed with excess of acetone and allowed to air dry overnight. Dialyzed against 10% NaCl solution for 3 days, followed by dialysis against pure water for 4 days. Freeze-dried to obtain the product.
- the degree of -carboxymethylation (D-CM) was 14% according to H 1 -NMR.
- methyl iodide (2.2 mL) was added followed by the drop wise addition of 2 eq. N,N-Diisopropylethylamine. After 20 hours of heating and refluxing the reaction mixture, it is allowed to room temperature, the product is precipitated with 200 mL EtOH, and then filtered on sintered funnel. The solid is washed with excess of acetone and allowed to air dry overnight. Dialyzed against 10% NaCl solution for 3 days, followed by dialysis against pure water for 4 days. Freeze-dried to obtain the product.
- the degree of N,N,N-trialkylation in the chitosan product was 33%, the N,N-dialkylation was 48% and the N-monoalkylation was 19% according to H 1 -NMR.
- the degree of 2-methoxyethoxy-ethyl was estimated 65%.
- the degree of N,N,N-trimethylation in the N-methylated chitosan product was 33% and N,N-dimethylation was 47% and N-monomethylation was 20% according to H 1 -NMR and O-methylation was not detected.
- N,N-Diisopropylethylamine N,N-Diisopropylethylamine. After 20 hours of heating and refluxing the reaction mixture, it is allowed to room temperature, the product is precipitated with 200 mL EtOH, and then filtered on sintered funnel. The solid is washed with excess of acetone and allowed to air dry overnight. Dialyzed against 10% NaCl solution for 3 days, followed by dialysis against pure water for 4 days. Freeze-dried to obtain the product.
- the degree of N,N,N-trialkylation in the N-methylated chitosan product was 20% and the N,N-dialkylation was 18% according to H 1 -NMR.
- the degree of hydroxypropyl trimethylation was found to be 37%.
- the degree of N,N,N-trimethylation in the N-methylated chitosan product was 26% and N,N-dimethylation was 34% and N-monomethylation was 40% according to H 1 -NMR.
- H-NMR spectroscopy was used to confirm the successful synthesis of TMC and to determine the degree of substitution.
- About 8-10 mg of each sample was dissolved in 0.8 mL D 2 O and a few drops of DCl and submitted for analysis.
- the tri-, di- and monomethyl peaks were observed at 3.3, 3.1 and 2.9 ppm, respectively (See FIG. 7 ; the shift for varied from 3.33 to 3.40 for the TM peak and from 3.06-3.12 for the DM peak, due to solvent effect) and the chitosan H2-H6 peaks of chitosan backbone were observed between 4.6-3.4 ppm (The N-acetyl peak at 2.08 was used as internal reference in the spectra).
- D-TM degree of N,N,N-trimethylation
- D-DM N,N-dimethylation
- D-MM N-monomethylation
- H-NMR spectroscopy was used to confirm the successful synthesis of N,N,N-triethylchitosan and to determine the degree of substitution.
- About 8-10 mg of each sample was dissolved in 0.8 mL D 2 O and a few drops of acetic acid d 4 and submitted for analysis.
- the received spectra were interpreted using Topspin software (Bruker).
- the peaks for the N-linked CH 2 groups of the alkyl chain where observed 3.40 ppm in case of N,N,N-trialkylation and at 3.27 in case of N,N-dialkylation, respectively and the chitosan H2-H6 peaks were observed between 4.6-3.4 ppm ( FIG. 5 ).
- D-TA degree of N,N,N-trialkylation
- D-DA N,N-dialkylation
- D - TA ⁇ ( CH 2 ) 3 6 ⁇ ( CH 2 ) 3 6 + ⁇ ( CH 2 ) 2 4 ⁇ 100 ⁇ %
- DDA ⁇ ( CH 2 ) 2 4 ⁇ ( CH 2 ) 3 6 + ⁇ ( CH 2 ) 2 4 ⁇ 100 ⁇ %
- the peak for tertiary methyl groups (methyl peaks for both N,N-dimethyl and N-alkyl, N-methyl substitution) could be observed at 3.08 ppm and the peak for quaternary methyl (methyl peaks for N,N,N-trimethyl, N-alkyl, N,N-dimethyl and N,N-dialkyl, N-methyl) could be observed at 3.34 ppm.
- H2-H6 peaks were observed between 4.6-3.4 ppm.
- the degree of N-hydroxypropylation (D-HP), tertiary N-methylation (D-TeM) and quaternary N-methylation (D-QM) were calculated from the integral values by the following equations: (the integrals for the protons of hydroxypropyl terminal methyl group, the quaternary ammonium methyl (as well as CH2 in the larger alkyl groups) and tertiary amine methyl (as well as CH2 larger alkyl groups) and protons H3-H6 (as well as proton H2 in acetylated monomers) indicated as ⁇ (CH 3 ) HP , ⁇ (CH 3 ) 3 and ⁇ (CH 3 ) 2 and ⁇ (H2-H6) respectively).
- H-NMR spectroscopy was used to confirm the successful synthesis of N-ethyl N-methyl chitosan and to determine the degree of substitution.
- About 8-10 mg of each sample was dissolved in 0.8 mL D 2 O and a few drops of DCl and submitted for analysis.
- the peaks for the terminal CH 3 groups of the N-linked ethyl chain can be observed at 1.38 ppm.
- the N-acetyl group can be observed at 2.07 ppm.
- the peak for tertiary methyl groups (methyl peaks for both N,N-dimethyl and N-alkyl, N-methyl substitution) could be observed at 3.08 ppm and the peak for quaternary methyl (methyl peaks for N,N,N-trimethyl, N-alkyl, N,N-dimethyl and N,N-dialkyl, N-methyl) could be observed at 3.34 ppm.
- H2-H6 peaks were observed between 4.6-3.4 ppm.
- the degree of N-ethylation (D-ET), quaternary N-methylation (D-QM and tertiary N-methylation (D-TeM)) were calculated from the integral values by the following equations: (the integrals for the ethyl terminal methyl group, the quaternary methyl groups and tertiary methyl groups and protons H3-H6 (as well as proton H2 in acetylated monomers) was indicated as ⁇ (CH 3 )Et, and ⁇ (CH 3 ) 3 , ⁇ (CH 3 ) 2 and ⁇ (H3-H6) respectively).
- the peak for tertiary methyl groups (methyl peaks for both N,N-dimethyl and N-alkyl, N-methyl substitution) could be observed at 3.08 ppm and the peak for quaternary methyl (methyl peaks for N,N,N-trimethyl, N-alkyl, N,N-dimethyl and N,N-dialkyl, N-methyl) could be observed at 3.37. ppm. H2-H6 peaks were observed between 4.6-3.4 ppm. and overlapped with the methoxy and ethoxy peaks.
- the degree of 2-methoxyethoxy-ethyl was estimated from the total integral of peaks in the range 4.6-3.4 ppm.
- H-NMR spectroscopy was used to confirm the successful synthesis of N-ethyl N-methyl chitosan and to determine the degree of substitution.
- About 8-10 mg of each sample was dissolved in 0.8 mL D 2 O and a few drops of DCl and submitted for analysis.
- the peaks for the quaternary N—CH 3 groups of the N-linked 2-hydroxypropyl-3-trimethyl ammonium moiety can be observed at 3.28 ppm.
- the N-acetyl group can be observed at 2.07 ppm.
- the peak for tertiary methyl groups (methyl peaks for both N,N-dimethyl and N-alkyl, N-methyl substitution) could be observed at 3.08 ppm and the peak for quaternary methyl (methyl peaks for N,N,N-trimethyl, N-alkyl, N,N-dimethyl and N,N-dialkyl, N-methyl) could be observed at 3.35 ppm.
- H2-H6 peaks were observed between 4.6-3.4 ppm.
- the degree of substitution N-hydroxypropyl 2-hydroxy)propyl-3-trimethyl ammonium (D-HPTM), tertiary N-methylation (D-TeM) and quaternary N-methylation (D-QM) were calculated from the integral values by the following equations: (the integrals for the HPTM trimethylgroup, quaternary methyl groups the tertiary methyl groups and was indicated as ⁇ (CH3)3-HPTM, ⁇ (CH3)3 and ⁇ (CH3) respectively).
- D - CM ⁇ ( CH 3 ) - CM 2 ⁇ ( H2 - H6 ) 6 ⁇ 100 ⁇ % .
Abstract
Description
-
- (a) dissolving chitosan, preferably in an acidic solution;
- (b) adding at least one suitable alkylation agent and at least one base in a water-miscible organic solvent,
-
- (a) dissolving a chitosan compound with formula
-
- wherein n is an integer greater than or equal to 3, wherein R is H and wherein W is —NH2 or N-acetyl,
- or a salt thereof, in a solution that contains at least 1 equivalent of an acid for each equivalent of —NH2 in the chitosan compound;
- (b) adding at least one alkylation agent selected from: (i) alkyl halides, dialkyl sulfates, dialkyl, alkyl methanosulfonates, carbonates, and alkyl-p-toluenesulfontes, with structure:
-
- and (ii) alkyl epoxides selected from:
-
- wherein
- n is an integer greater than or equal to 3,
-
- 1. Novel N-alkylated chitosan derivatives that are water soluble and therefore particularly useful in a range of applications.
- 2. A one-pot synthetic process of N-alkylated chitosan, including trimethylchitosan, not requiring the isolation of intermediate compounds.
- 3. Minimal O-alkylation in the resulting product compound.
- 4. Use of a small excess of the alkylation agent, thus resulting in reduced cost of synthesis
- 5. Relatively short reaction time (hours not many days)
- 6. Synthetic protocol that is easily scalable for industrial production.
- 7. The reaction can be repeated to obtain very high degree of trialkylation with minimal O-alkylation.
- 8. Alkylation can be directed by repeating alkylation reaction and/or using a mixture of alkylating agents.
Claims (12)
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EP0249779A1 (en) | 1986-06-16 | 1987-12-23 | Etablissement Texcontor | Methylated chitosans and their use for the preparation of pharmaceutical compositions |
US4772690A (en) | 1985-01-29 | 1988-09-20 | Wella Aktiengesellschaft | Quaternary hydroxyethyl-substituted chitosan derivatives, cosmetic compositions based thereon and processes for the production thereof |
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-
2019
- 2019-08-23 CA CA3110181A patent/CA3110181A1/en active Pending
- 2019-08-23 WO PCT/IS2019/050009 patent/WO2020039463A1/en unknown
- 2019-08-23 US US17/270,237 patent/US11535684B2/en active Active
- 2019-08-23 EP EP19783736.2A patent/EP3841129A1/en active Pending
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